Lentiviral vector-based vaccine

ABSTRACT

Methods of eliciting humoral responses, methods of immunization, and methods of vaccination using lentiviral vector are disclosed. Additionally, immunogenic compositions and vaccines for West Nile Virus are disclosed.

FIELD OF THE INVENTION

The invention is directed to lentiviral vector, methods of genetransfer, immunization, and vaccination using lentiviral vector,processes for the production of recombinant polypeptides, antibodiesgenerated against these polypeptides, and the use of such molecules indiagnostic methods, kits, immunogenic compositions, vaccines, andantiviral therapy.

BACKGROUND OF THE INVENTION

In the past few years, lentiviral gene transfer vectors have gained aconsiderable interest among the gene therapy community. This unmatchedreputation is due to their ability to efficiently and stably transfertherapeutic or reporter genes to a large variety of cells and tissues ofkey importance for therapeutic intervention, such as hematopoietic stemcells, brain, liver and retina (see [1-4], among others). Lentiviralvectors achieve high transduction efficiency irrespective of theproliferative status of the target cells, thus circumventing one of themain limitations of oncovirus-derived retroviral vectors in whichtransduction is restricted to dividing cells.

This advantage is reflected in a number of successful preclinical testsin various animal models of human diseases (see [5-11], among others),and will undoubtedly translate into their exploitation in a growingnumber of clinical trials.

More recently, lentiviral vectors have also been proven as promisingvaccination vectors. Most studies to date have focused on the inductionof cellular immune responses in the field of anti-tumoral immunotherapy[12-18], a few have also focused on the induction of protective cellularimmunity against viruses [19-21].

Their capacity to transduce non-dividing dendritic cells (DCs) with highefficiency, ex-vivo [12, 14, 15, 19] as well as in vivo [13, 18, 22],accounts for their ability to elicit strong CTL responses.

Indeed, reports using lentiviral vectors to stimulate anti-tumorimmunity show very promising results. Human DCs transduced by lentiviralvectors expressing tumor antigens stimulate specific CTL responses invitro [12, 14, 15, 18]. In mice, the injection of lentiviral vectorparticles or lentiviral vector transduced DCs induce strong and specificanti-tumor cellular immune responses [13, 15, 18] and confer protectionfrom tumor challenge [12, 17]. These responses are more potent than theones obtained by classical immunization with peptide plus adjuvant [13]or even peptide-pulsed antigen presenting cells (APCs) whether assayedex vivo [20] or in vivo [16, 21]. The observed advantage could be due tothe continuous presentation of the antigen in vector-transduced APCs incontrast to the transient antigen presentation that ensues from peptidepulsing of APCs [16, 20].

To date, little is known of the ability of lentiviral vectors tostimulate an antibody-based protective immunity. Nevertheless, theirhigh transduction efficiency could also grant them a strong ability toinduce humoral immunity. Moreover, their seemingly preferential tropismfor DCs when injected in vivo [13, 18, 22] could further enhance theircapacity to elicit a protective antibody-based immunity, since DCs arepowerful stimulators of CD4 T cells, which are needed for the correctdevelopment of a B-cell based immune response. Furthermore, in avaccination scenario, the stable expression of the antigen fromtransduced cells might preclude the need for several injections.

Indeed, it is widely accepted that the humoral immune response is theessential component of protective immunity against WNV [23-25]. Theenvelope E-glycoprotein from WNV, which possesses neutralizing epitopes[26], elicits protective immune responses when injected as a recombinantantigen [27] or expressed either by naked DNA [28] or a replicativemeasles vector [29]. The fact that passive transfer of neutralizingantibodies to the soluble form of the envelope E glycoprotein (sE) fromWNV strain IS-98-ST1 protected mice from WNV encephalitis furtherdemonstrates that the humoral response is sufficient for protectionagainst WNV challenge [29].

WNV is a mosquito-borne flavivirus in the Japanese encephalitisserocomplex of the Flaviviridae family. It is transmitted in naturalcycles between birds and mosquitoes, but it can infect many species ofmammals [30]. Zoonotic WNV recently became a major health concern inNorth America, the Middle East, and Europe, due to the emergence of amore virulent strain in Israel in 1998 and then in New York in 1999(Isr98/NY99). Severe clinical manifestations of WNV infectionessentially involve the central nervous system and can cause significantmortality in humans and in a large range of animal species, particularlyhorses and birds (for review see [30]). As a consequence, an urgentdemand exists for the development of an efficient vaccine that canprovide quick, strong and long lasting immunity.

SUMMARY OF THE INVENTION

This invention aids in fulfilling these needs in the art. This inventionrelates to the potential of lentiviral vectors to elicit a humoralimmune response. Whether immunization with a lentiviral-based vaccinecould protect against West Nile Virus (WNV) infection was investigated.It was surprisingly discovered that lentiviral vectors can elicitantibody based protective immunity.

In the present invention, the potential of a lentiviral vector-basedvaccine was evaluated to elicit humoral immunity against West Nile Virus(WNV), a mosquito-borne flavivirus that causes encephalitis in humans,birds and horses. Remarkably, a single immunization with a minute doseof TRIP/sE_(WNV), a lentiviral vector expressing the secreted solubleform of the envelope E-glycoprotein (sE_(WNV)) from the highly virulentIS-98-ST1 strain of WNV, induced a specific humoral response andprotection against WNV infection in a mouse model of WNV encephalitis.This single immunization elicited a long-lasting, protective andsterilizing humoral immunity, only one week after priming. These resultsdemonstrate the applicability of lentiviral vectors as efficientnon-replicating vaccines against pathogens for which a neutralizinghumoral response is actively required for protective immunity.TRIP/sE_(WNV) lentiviral vector appears as a promising tool forveterinary vaccination against zoonotic WNV.

Thus, this invention provides a method of producing antibodies in vivocomprising administration of a lentiviral vector to an animal, whereinthe lentivirus comprises a heterologous nucleic acid encoding anantigen, and wherein the antigen elicits a humoral response in theanimal. The invention encompasses a lentiviral vector that inducesprotective immunity to an animal in need thereof, including, forexample, long-lasting protective immunity and sterilizing immunity. Inone embodiment, the animal in need thereof is selected from humans;horses; birds; poultry; pigs; cattle, including bovines, ovins, andcaprins; rodents, including hamsters, rats, and mice; pets; andreptiles.

This invention additionally provides a method of producing antibodies invivo comprising administration of a lentiviral vector to an animal,wherein the lentivirus comprises a heterologous nucleic acid encoding anantigen, and wherein the antigen elicits a humoral response in theanimal, wherein the lentiviral vector induces protective immunityagainst an infectious microorganism In one embodiment, the infectiousmicroorganism is West Nile Virus. In another embodiment, the lentiviralvector comprises a nucleic acid encoding a peptide or polypeptidebearing at least a B epitope. The polypeptide bearing at least a Bepitope can be, for example, a microorganism membrane protein or afragment thereof, and in particular envelope E-glycoprotein from WestNile Virus, or a fragment thereof. The lentiviral vector may comprise acarboxyl terminal-truncated E glycoprotein from West Nile Virus lackingthe transmembrane anchoring region, for example, a carboxylterminal-truncated E glycoprotein comprising residues 1-441 of theenvelope E-glycoprotein from West Nile Virus strain IS-98-ST1. Inanother embodiment, the heterologous nucleic acid encodes a variant ofenvelope E-glycoprotein from West Nile Virus. The invention encompassesa variant heterologous nucleic acid that hybridizes under conditions ofstringent hybridization, which are described hereinafter, to a nucleicacid from West Nile Virus strain IS-98-ST1 encoding envelopeE-glycoprotein.

The invention also provides for a method for vaccinating an animalagainst West Nile Virus infection, comprising administering to an animalin need thereof, one or more times, a lentiviral vector comprising anucleic acid encoding a peptide bearing at least a B epitope. Thepeptide bearing at least a B epitope can be, for example, amicroorganism membrane protein or a fragment thereof, and in particularenvelope E-glycoprotein from West Nile Virus, or a fragment thereof,with an acceptable physiological carrier and/or an adjuvant. In vivosuitable routes of administration include, for example, anintraperitoneal route, intravenous route, intramuscular route, oralroute, mucosal route, sublingual route, intranasal route, subcutaneousroute or intradermic route. Ex vivo suitable routes includeadministration of autologous cells transduced with the lentiviral vector[i.e. Antigen Presenting Cells (APC) as dendritic cells (DC) or Bcells]. In one embodiment, the heterologous nucleic acid encodes apeptide or polypeptide bearing at least a B epitope. In a preferredembodiment, the polypeptide encodes a membrane protein or a fragmentthereof. In another preferred embodiment, the heterologous nucleic acidencodes carboxyl terminal-truncated E glycoprotein from West Nile Viruslacking the transmembrane anchoring region. In another, the carboxylterminal-truncated E glycoprotein comprises residues 1-441 of theenvelope E-glycoprotein from West Nile Virus strain IS-98-ST1. Apreferred embodiment of this invention encompasses administering thelentiviral vector in a limited number of doses, such as, for example, asingle dose or two or three doses. In one embodiment, this dosecomprises, for example, vector particles equivalent to 0.5 ng to 5000 ngof p24 antigen. In mice, the preferred dose can vary from 0.5 ng to 50ng, while in horses the preferred dose can vary from 50 ng to 500 ng.The invention encompasses the treatment of such animals, for example, ashumans; horses; birds; poultry; pigs; cattle, including bovines, ovins,and caprins; rodents, including hamsters, rats, and mice; pets; andreptiles.

The invention also provides for a method for vaccinating an animalagainst microorganism infection, comprising administering to an animalin need thereof, one or more times, a lentiviral vector comprising aheterologous nucleic acid encoding an immunogenic membrane protein or afragment thereof of such microorganism. As used herein, the termmicroorganism encompasses viruses, bacteria, and parasites.

In a preferred embodiment the invention provides a method forvaccinating an animal in need thereof against virus infection, such as aflavivirus infection, and in particular a method for vaccinating ananimal in need thereof against West Nile Virus infection byadministering a lentiviral vector comprising a variant of envelopeE-glycoprotein from West Nile Virus, or a fragment thereof, with anacceptable physiological carrier and/or an adjuvant. The inventionencompasses such a method of vaccination wherein the heterologousnucleic acid hybridizes under conditions of stringent hybridization to anucleic acid from West Nile Virus strain IS-98-ST1 encoding envelopeE-glycoprotein. In one embodiment, the lentiviral vector is administeredone time, for example, in a dose of vector particles equivalent to 0.5ng to 5000 ng of p24 antigen. The animal in need thereof can include,for example, humans; horses; birds; poultry; pigs; cattle, includingbovines, ovines, and caprines; rodents, including hamsters, rats, andmice; pets, such as cats and dogs; and reptiles.

The invention also provides for an immunogenic composition comprising alentiviral vector, wherein the lentiviral vector comprises aheterologous nucleic acid encoding an antigen in an amount sufficient toinduce an immunogenic or protective response in vivo, and apharmaceutically acceptable carrier therefor. The invention encompassesan immunogenic composition, wherein the heterologous nucleic acidencodes a peptide bearing at least a B epitope. A peptide bearing atleast a B epitope can be, for example, a microorganism membrane proteinor a fragment thereof, and in particular envelope E-glycoprotein fromWest Nile Virus, or a fragment thereof, or a carboxyl terminal-truncatedE-glycoprotein from West Nile Virus lacking the transmembrane anchoringregion. In one embodiment, the carboxyl terminal-truncated Eglycoprotein comprises residues 1-441 of the envelope E-glycoproteinfrom West Nile Virus strain IS-98-ST1.

The invention also provides for an immunogenic composition comprising alentiviral vector, wherein the lentiviral vector comprises aheterologous nucleic acid encoding an antigen in an amount sufficient toinduce an immunogenic or protective response in vivo, and apharmaceutically acceptable carrier therefor, and wherein theheterologous nucleic acid encodes a variant of envelope E-glycoproteinfrom West Nile Virus. In one example, the heterologous nucleic acidhybridizes under conditions of stringent hybridization to a nucleic acidfrom West Nile Virus strain IS-98-ST1 encoding envelope E-glycoprotein.

The invention encompasses a lentiviral vector that directs theexpression of a heterologous nucleic acid, wherein the vector comprisescytomegalovirus immediate early promoter, and wherein the heterologousnucleic acid encodes envelope E-glycoprotein from West Nile Virus, or afragment thereof. The heterologous nucleic acid can encode, for example,carboxyl terminal-truncated E-glycoprotein from West Nile Virus lackingthe transmembrane anchoring region. In one embodiment, the carboxylterminal-truncated E-glycoprotein comprises residues 1-441 of envelopeE-glycoprotein from West Nile Virus strain IS-98-ST1. The invention alsoprovides for a substantially purified host cell transfected ortransduced with such a lentiviral vector, and for a method for theproduction of envelope E-glycoprotein polypeptide from West Nile Virus,or a fragment thereof, comprising culturing a host cell transfected ortransduced with such a lentiviral vector under conditions promotingexpression, and recovering the polypeptide from the host cell or theculture medium.

The invention further provides for a lentiviral vector that directs theexpression of a heterologous nucleic acid, wherein the vector comprisescytomegalovirus immediate early promoter, and wherein the heterologousnucleic acid encodes a variant of envelope E-glycoprotein from West NileVirus. In one embodiment, the heterologous nucleic acid hybridizes underconditions of stringent hybridization to a nucleic acid from West NileVirus strain IS-98-ST1 encoding envelope E-glycoprotein. The inventionencompasses a substantially purified host cell transfected or transducedwith such a lentiviral vector, and for a method for the production of avariant of envelope E-glycoprotein from West Nile Virus comprisingculturing a host cell transfected or transduced with such a lentiviralvector under conditions promoting expression, and recovering thepolypeptide from the host cell or the culture medium.

The invention further provides for a lentiviral vector that directs theexpression of a heterologous nucleic acid, wherein the vector comprisescytomegalovirus immediate early promoter, and wherein the heterologousnucleic acid comprises green fluorescent protein. The invention alsoencompasses a substantially purified host cell transfected or transducedwith this lentiviral vector.

The invention encompasses a method of vaccinating against a pathogenicagent comprising administering a lentiviral vector to an animal in needthereof, wherein the lentiviral vector comprises a heterologous nucleicacid encoding an antigen, and wherein the antigen elicits antibodiesagainst the pathogenic agent. The invention encompasses, for example, amethod of vaccinating wherein administration of the lentiviral vectorelicits long-lasting humoral immunity. The invention also encompasses,for example, a method of vaccinating wherein administration of thelentiviral vector elicits neutralizing antibodies against the pathogenicagent. The animal in need thereof can include, for example, humans;horses; birds; poultry; pigs; cattle, including bovines, ovines, andcaprines; rodents, including hamsters, rats, and mice; pets, such ascats and dogs; and reptiles. In one embodiment, the lentiviral vectorcomprises a nucleic acid encoding envelope E-glycoprotein from West NileVirus, or a fragment thereof, and the pathogenic agent is West NileVirus. In another embodiment, the envelope E-glycoprotein from West NileVirus lacks a transmembrane anchoring region. In another, theadministration of lentiviral vector comprises at least one dose ofvector particles equivalent to 0.5 ng to 5000 ng of p24 antigen.

The invention also provides for a method of delivering DNA for producingantibodies in vivo comprising administration of a lentiviral vector toan animal, wherein the lentiviral vector comprises a heterologousnucleic acid encoding an antigen, and wherein the antigen elicits ahumoral response in the animal. The invention encompasses a lentiviralvector that induces protective humoral immunity to an animal in needthereof. In one embodiment, the animal in need thereof is selected fromhumans; horses; birds; poultry; pigs; cattle, including bovines, ovins,and caprins; rodents, including hamsters, rats, and mice; pets; andreptiles.

This invention additionally provides for a method of delivering DNA forproducing antibodies in vivo comprising administration of a lentiviralvector to an animal, wherein the lentiviral vector comprises aheterologous nucleic acid encoding an antigen, and wherein the antigenelicits a humoral response in the animal, wherein the lentiviral vectorinduces protective immunity against West Nile Virus. In one embodiment,the lentiviral vector may comprise a nucleic acid encoding an envelopeE-glycoprotein from West Nile Virus, or a fragment thereof. Thelentiviral vector may comprise a nucleic acid encoding a carboxylterminal-truncated E glycoprotein from West Nile Virus lacking thetransmembrane anchoring region, for example, a carboxylterminal-truncated E glycoprotein comprising residues 1-441 of theenvelope E-glycoprotein from West Nile Virus strain IS-98-ST1. Inanother embodiment, the heterologous nucleic acid encodes a variant ofenvelope E-glycoprotein from West Nile Virus. The invention encompassesa variant heterologous nucleic acid that hybridizes under conditions ofstringent hybridization, which are described hereinafter, to a nucleicacid from West Nile Virus strain IS-98-ST1 encoding envelopeE-glycoprotein.

The invention also encompasses a method comprising administration of alentiviral vector to an animal, wherein the lentiviral vector comprisesa heterologous nucleic acid encoding an antigen, and wherein the antigenelicits a humoral response in the animal. The invention encompasses alentiviral vector that induces protective humoral immunity to an animalin need thereof. In one embodiment, the animal in need thereof isselected from humans; horses; birds; poultry; pigs; cattle, includingbovines, ovins, and caprins; rodents, including hamsters, rats, andmice; pets; and reptiles.

The invention additionally provides for a method comprisingadministration of a lentiviral vector to an animal, wherein thelentiviral vector comprises a heterologous nucleic acid encoding anantigen, wherein the antigen elicits a humoral response in the animal,and wherein the antigen expressed in the animal after their transductionwith the lentiviral vector induces protective immunity against aninfectious microorganism, such as, for example, West Nile Virus. In oneembodiment, the lentiviral vector comprises a nucleic acid encoding amembrane protein from an infectious microorganism (in particularencoding an envelop protein and more preferentially, the envelopeE-glycoprotein from West Nile Virus, or a fragment thereof. Thelentiviral vector may comprise a nucleic acid encoding the carboxylterminal-truncated E glycoprotein from West Nile Virus lacking thetransmembrane anchoring region, for example, a carboxylterminal-truncated E glycoprotein comprising residues 1-441 of theenvelope E-glycoprotein from West Nile Virus strain IS-98-ST1. Inanother embodiment, the heterologous nucleic acid encodes a variant ofenvelope E-glycoprotein from West Nile Virus. In one embodiment, theinvention encompasses a heterologous nucleic acid that hybridizes underconditions of stringent hybridization to those described hereinafter,for example, to a nucleic acid from West Nile Virus strain IS-98-ST1encoding envelope E-glycoprotein.

Additional objects and advantages of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Theobjects and advantages of the invention will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the Expression of sE_(WNV) in 293T cells transduced withTRIP/sE_(WNV). (A) Schematic representation of the TRIP/sE_(WNV) vector.The IS-98-ST1 cDNA coding for sE_(WNV) was subcloned from theTOPO/sE_(WNV) plasmid [29] into the TRIP lentiviral vector between theBsiW1 and BssHII cloning sites and under the control of the humancytomegalovirus immediate early promoter (CMVie). LTR=Long TerminalRepeat. (B) Detection of sE_(WNV) protein in TRIP/sE_(WNV) transduced293T cells. 293T cells were transduced with the amount of TRIP/sE_(WNV)vector particles equivalent of 100 ng/ml of p24 antigen (left), or leftuntransduced (right). At 48 hours post-transduction, 293T cells wereimmunostained with an anti-WNV HMAF (Hyperimmune Mouse Ascitic Fluid).

FIG. 2 shows the detection of anti-E_(WNV) antibodies in sera fromTRIP/sE_(WNV) vaccinated mice. Vero cells were infected with WNV strainIS-98-ST1 (WNV) or mock-infected (No virus). Radio-labeled cell lysateswere immunoprecipitated with pooled immune sera (dilution 1:100).Samples were analyzed by SDS-15% PAGE under non-reducing conditions. (A)Sera from TRIP/sE_(WNV) immunized mice collected at day 6, 13, 20 and 27post-immunization (p.i). (B) Sera from resistant congenic BALB/c-MBTmice inoculated with WNV (sera to WNV). Antisera tolymphochoriomeningitis virus (LCMV) were used as a negative control. WNVstructural glycoproteins C, prM and E and non-structural proteins NS3and NS2A are shown. (C) Sera from TRIP/sE_(WNV) immunized mice collected21 days after WNV challenge, which was performed 7 or 14 dayspost-immunization. As a positive control, viral antigens wereimmunoprecipitated with anti-WNV HMAF.

FIG. 3 shows that heat-inactivation of TRIP/sE_(WNV) vector particlesabolishes transduction. 293T cells were transduced with a TRIP/GFPvector (66 ng of p24 antigen per ml), heat-inactivated (70° C. for 10min) or not. At 48 h post-transduction, GFP expression was detected byFACS.

FIG. 4 shows the nucleic acid sequence of the secreted form of the WestNile Virus E-glycoprotein from strain IS-98-ST1, with start and stopcodons added at the ends of the sequence.

FIG. 5 shows the nucleic acid sequence of the secreted form of the WestNile Virus E-glycoprotein from strain IS-98-ST1.

FIG. 6 shows the amino acid sequence of the secreted form of the WestNile Virus E-glycoprotein from strain IS-98-ST1. The signal sequence atthe N-terminus is underlined.

FIG. 7 shows the amino acid sequence of Domain III from West Nile VirusE-glycoprotein from strain IS-98-ST1.

FIG. 8 shows the complete nucleic acid sequence of the lentiviral vectorpTRIPsE_(WNV) bearing the heterologous sequence of truncated West NileVirus E-glycoprotein from West Nile Virus strain IS-98-ST1.

DETAILED DESCRIPTION

Lentiviral vectors for inducing protective humoral responses have beendiscovered. In one embodiment, the lentiviral vectors arereplication-defective retroviral vectors comprising cis-active sequencesnecessary for reverse transcription (PPT 3′, PBS and R regions of theLTRs), nuclear import (cPPT-CTS), and integration (psi), and aheterologous nucleic acid encoding an antigen. In this embodiment,structural and enzymatic proteins are provided in “trans.” Thelentiviral vectors can comprise integration repeat (IR) sequences andTips of the LTR, as well as transcriptional sequences as ORF andpromoters. The lentiviral vector particle can be pseudotyped by anyviral or non-viral envelope to facilitate its entry into the targetcell. One preferred site on the lentiviral vector for insertion of theheterologous nucleic acid is between the two LTRs, as explained below.Alternately, the heterologous nucleic acid can be, for example, insertedinto the U3 or U5 regions of the LTRs, or provided as a replacement forthe U3 or U5 regions of the LTRs.

In one embodiment, the lentiviral vector comprises a heterologousnucleic acid encoding a peptide bearing at least one B epitope. Inpreferred embodiments, the heterologous nucleic acid encodes a membraneprotein from an infectious microorganism, such as, for example, anenvelope protein. In one preferred embodiment, the heterologous nucleicacid encodes envelope E-glycoprotein from West Nile Virus, or a fragmentthereof.

The term “lentiviral vector” means that the vector contains apolynucleotide sequence that: (1) is not associated with all or aportion of a polynucleotide with which it is associated in nature, (2)is linked to a polynucleotide other than that to which it is linked innature, or (3) does not occur in nature. Lentiviral vectors of theinvention encompass vectors derived from, for example, HIV-1, HIV-2, SIV(simian immunodeficiency virus), EIAV (equine infectious anaemia virus),FIV (feline immunodeficiency virus), CAEV (Caprine arthritisencephalitis virus), and VMV (Visna/maedi virus). Lentiviral vectorsalso encompass chimeric lentiviruses derived from at least two differentlentiviruses.

The terms “polypeptide” and “protein”, used interchangeably herein,refer to a polymeric form of amino acids of any length, and includeschemically or biochemically modified or derivatized amino acids, as wellas polypeptides having modified peptide backbones. The term includesfusion proteins, such as GST fusion proteins and pegylated proteins,fusion proteins with a heterologous amino acid sequence, fusions withheterologous or homologous leader sequences, with or without N-terminalmethionine residues; immunologically tagged proteins; and the like.

A “fragment” of a polypeptide sequence refers to a polypeptide sequencethat is shorter than the reference sequence but that retains abiological function or activity which is recognized to be the same asthe reference polypeptide. Such an activity may include, for example,the ability to stimulate an immune response. A fragment retains at leastone epitope of the reference polypeptide.

The term “purified” as used herein, means that a polypeptide that isessentially free of association with other proteins or polypeptides, forexample, as a purification product of recombinant host cell culture oras a purified product from a non-recombinant source. The term“substantially purified”, as used herein, refers to a mixture thatcontains a polypeptide and is essentially free of association with otherproteins or polypeptides, but for the presence of known proteins thatcan be removed using a specific antibody, and which substantiallypurified polypeptides can be used as an antigen.

The term “antigen” as used herein, means a substance capable ofstimulating an immune response. Preferred antigens encompass at leastone B-epitope, wherein a B-epitope is capable of eliciting a humoralimmune response. Such preferred antigens include, for example, surfaceantigens, such as envelope or other membrane proteins, and fragmentsthereof.

The term “pathogen” as used herein, means a specific causative agent ofdisease, and may include, for example, any bacteria, virus or parasite.

The term “disease” as used herein, means an interruption, cessation, ordisorder of body function, system, or organ. Preferred diseases includeinfectious diseases.

The term “humoral immunity” as used herein, means antibodies elicited byan antigen, and all the accessory processes that accompany it.

The term “protective humoral immunity” as used herein, means a humoralimmune response that confers the essential component of protectionagainst a pathogen.

The term “sterilizing humoral immunity” as used herein, means a humoralimmune response that prevents the establishment of any detectableinfection by a pathogen.

The term “long-lasting humoral immunity” as used herein, means that someaspect of humoral immunity is detectable three months after antigenadministration, such as, for example, antibodies elicited by theantigen. Suitable methods of antibody detection include, but are notlimited to, such methods as ELISA, immunofluorescence (IFA), focusreduction neutralization tests (FRNT), immunoprecipitation, and Westernblotting.

The term “neutralizing humoral response” as used herein, means that theantibodies elicited during humoral immunity directly block the abilityof a pathogen to infect cells.

The term “quick response” as used herein, means that protective humoralimmunity is conferred within three weeks of antigen administration. Theterm “very quick response” as used herein, means that protective humoralimmunity is conferred within one week of antigen administration.

Hybridization reactions can be performed under conditions of different“stringency.” Conditions that increase stringency of a hybridizationreaction are known in the art. For example, stringent conditions forboth DNA/DNA and DNA/RNA hybridization are described in Sambrook et al.Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. Moreover, a personskilled in the art would know how to modify the conditions as necessaryfor the degree of stringency required for a particular hybridization.

Examples of relevant conditions include (in order of increasingstringency): incubation temperatures of 25° C., 37° C., 50° C. and 68°C.; buffer concentrations of 10×SSC, 6×SSC, 1×SSC, 0.1×SSC (where 1×SSCis 0.15 M NaCl and 15 mM citrate buffer) and their equivalents usingother buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%;incubation times from 5 minutes to 24 hours; 1, 2, or more washingsteps; wash incubation times of 1, 2, or 15 minutes; and wash solutionsof 6×SSC, 1×SSC, 0.1×SSC, or deionized water.

An example of stringent hybridization conditions is hybridization at 50°C. and 0.1×SSC (15 mM sodium chloride/1.5 mM sodium citrate). Anotherexample of stringent hybridization conditions is overnight incubation at42° C. in a solution containing 50% formamide, 1×SSC (150 mM NaCl, 15 mMsodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution,10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 0.1×SSC at about 65° C. A furtherexample of high stringency conditions includes aqueous hybridization(e.g., free of formamide) in 6×SSC (where 20×SSC contains 3.0 M NaCl and0.3 M sodium citrate), 1% sodium dodecyl sulfate (SDS) at 65° C. forabout 8 hours (or more), followed by one or more washes in 0.2×SSC, 0.1%SDS at 65° C.

A “variant” polypeptide as referred to herein means a polypeptidesubstantially homologous to the reference polypeptide, but which has anamino acid sequence different from that of reference polypeptidesbecause of one or more deletions, insertions, or substitutions. Thevariant amino acid sequence preferably is at least 90% identical to thereference polypeptide, and most preferably at least 95% identical. Thepercent identity can be determined, for example by comparing sequenceinformation using the GAP computer program, version 6.0 described byDevereux et al. (Nucl. Acids Res. 12:387, 1984) and available from theUniversity of Wisconsin Genetics Computer Group (UWGCG). The GAP programutilizes the alignment method of Needleman and Wunsch (J. Mol. Biol.48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math 2:482,1981). The preferred default parameters for the GAP program include: (1)a unary comparison matrix (containing a value of 1 for identities and 0for non-identities) for nucleotides, and the weighted comparison matrixof Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described bySchwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure,National Biomedical Research Foundation, pp. 353-358, 1979; (2) apenalty of 3.0 for each gap and an additional 0.10 penalty for eachsymbol in each gap; and (3) no penalty for end gaps.

Variants can comprise conservatively substituted sequences, meaning thata given amino acid residue is replaced by a residue having similarphysiochemical characteristics. Examples of conservative substitutionsinclude substitution of one aliphatic residue for another, such as Ile,Val, Leu, or Ala for one another, or substitutions of one polar residuefor another, such as between Lys and Arg; Glu and Asp; or Gln and Asn.Other such conservative substitutions, for example, substitutions ofentire regions having similar hydrophobicity characteristics, are wellknown. Naturally occurring polypeptide variants are also encompassed bythe invention. Examples of such variants are proteins that result fromalternate mRNA splicing events or from proteolytic cleavage of thepolypeptides. Variations attributable to proteolysis include, forexample, differences in the termini upon expression in different typesof host cells, due to proteolytic removal of one or more terminal aminoacids from the polypeptides. Variations attributable to frameshiftinginclude, for example, differences in the termini upon expression indifferent types of host cells due to different amino acids.

The term “binds specifically,” in the context of antibody binding,refers to high avidity and/or high affinity binding of an antibody to aspecific polypeptide, or more accurately, to a specific epitope of aspecific polypeptide. Antibody binding to such epitope is typicallystronger than binding of the same antibody to any other epitope or anyother polypeptide which does not comprise the epitope. Such an antibodyis typically produced by injecting the specific polypeptide into ananimal to elicit the production of antibodies. Such an antibody may becapable of binding other polypeptides at a weak, yet detectable, level(e.g., 10% or less of the binding shown to the polypeptide of interest).Such weak binding, or background binding, is readily discernible fromthe specific antibody binding, for example, by use of appropriatecontrols. In general, antibodies of the invention specifically bind to aspecific polypeptide with a binding affinity of 10⁻⁷ M or more,preferably 10⁻⁸ M or more (e.g., 10⁻⁹ M, 10⁻¹⁰, 10⁻¹¹, etc.).

A “pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any conventional type. A “pharmaceuticallyacceptable carrier” is non-toxic to recipients at the dosages andconcentrations employed and is compatible with other ingredients of theformulation. For example, the carrier for a formulation containingpolypeptides preferably does not include oxidizing agents and othercompounds that are known to be deleterious to polypeptides. Suitablecarriers include, but are not limited to, water, dextrose, glycerol,saline, ethanol, and combinations thereof. The carrier may containadditional agents such as wetting or emulsifying agents, pH bufferingagents, or adjuvants which enhance the effectiveness of the formulation.Topical carriers include liquid petroleum, isopropyl palmitate,polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) inwater, or sodium lauryl sulfate (5%) in water. Other materials such asanti-oxidants, humectants, viscosity stabilizers, and similar agents maybe added as necessary. Percutaneous penetration enhancers such as Azonemay also be included.

The invention provides for isolated and purified, or homogeneous,envelope E-glycoprotein polypeptides from West Nile Virus, bothrecombinant and non-recombinant. Variants and derivatives of nativeenvelope E-glycoprotein polypeptides that can be used as antigens can beobtained by mutations of nucleotide sequences coding for native envelopeE-glycoprotein polypeptides. Alterations of the native amino acidsequence can be accomplished by any of a number of conventional methods.Mutations can be introduced at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene whereinpredetermined codons can be altered by substitution, deletion, orinsertion. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981);Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methodsin Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462,all of which are incorporated by reference.

Within an aspect of the invention, the lentiviral vector can be utilizedto prepare antibodies that specifically bind to polypeptides. The term“antibodies” is meant to include polyclonal antibodies, monoclonalantibodies, fragments thereof such as F(ab′)2 and Fab fragments, as wellas any recombinantly produced binding partners. Antibodies are definedto be specifically binding if they bind envelope E-glycoproteinpolypeptides with a K_(a) of greater than or equal to about 10⁷ M⁻¹.Affinities of binding partners or antibodies can be readily determinedusing conventional techniques, for example, those described by Scatchardet al., Ann. N.Y Acad. Sci., 51:660 (1949). Polyclonal antibodies can bereadily generated from a variety of sources, for example, horses, cows,goats, sheep, dogs, chickens, rabbits, mice, or rats, using proceduresthat are well known in the art.

Recombinant expression vectors can be prepared using well known methods.For a review of molecular biology techniques see Sambrook, et al.Molecular Cloning: A Laboratory Manual, CSH Press 1989. The expressionvectors can include an insert sequence, such as West Nile Virus envelopeE-glycoprotein sequence, operably linked to suitable transcriptional ortranslational regulatory nucleotide sequences, such as those derivedfrom a mammalian, microbial, viral, or insect gene. Examples ofregulatory sequences include transcriptional promoters, operators, orenhancers, an mRNA ribosomal binding site, and appropriate sequenceswhich control transcription and translation initiation and termination.Nucleotide sequences are “operably linked” when the regulatory sequencefunctionally relates to the insert sequence. Thus, a promoter nucleotidesequence is operably linked to a envelope E-glycoprotein sequence if thepromoter nucleotide sequence controls the transcription of the envelopeE-glycoprotein DNA sequence. The ability to replicate in the desiredhost cells, usually conferred by an origin or replication, and aselection gene by which transformants are identified can additionally beincorporated into the expression vector.

In addition, sequences encoding appropriate signal peptides that are notnaturally associated with the insert sequence can be incorporated intoexpression vectors.

Suitable host cells for expression include prokaryotes, yeast or highereukaryotic cells. Appropriate cloning and expression vectors for usewith bacterial, fungal, yeast, and mammalian cellular hosts aredescribed, for example, in Pouwels et al. Cloning Vectors: A LaboratoryManual, Elsevier, N.Y., (1985).

Once the lentiviral vectors of the invention have been obtained, theycan be used to produce polyclonal and monoclonal antibodies. Thus, aprotein or polypeptide expressed in vivo or ex vivo by means of thelentiviral vector of the invention can be used to immunize an animalhost by techniques known in the art. Such techniques usually involveinoculation, but they may involve other modes of administration. Asufficient amount of the lentiviral vector is administered to create animmunogenic response in the animal host. Any host that can be infectedby lentivirus can be used. Once the animal as been immunized andsufficient time has passed for it to begin producing antibodies to theantigen, polyclonal antibodies can be recovered. The general methodcomprises removing blood from the animal and separating the serum fromthe blood. The serum, which contains antibodies to the antigen, can beused as an antiserum to the antigen. Alternatively, the antibodies canbe recovered from the serum. Affinity purification is a preferredtechnique for recovering purified polyclonal antibodies to the antigen,from the serum.

Monoclonal antibodies to the antigens of the invention can also beprepared. One method for producing monoclonal antibodies reactive withthe antigens comprises the steps of infecting the host with thelentiviral vector; recovering antibody producing cells from the spleenof the host; fusing the antibody producing cells with myeloma cellsdeficient in the enzyme hypoxanthine-guanine phosphoribosyl transferaseto form hybridomas; selecting at least one of the hybridomas by growthin a medium comprising hypoxanthine, aminopterin, and thymidine;identifying at least one of the hybridomas that produces an antibody tothe antigen, culturing the identified hybridoma to produce antibody in arecoverable quantity; and recovering the antibodies produced by thecultured hybridoma.

These polyclonal or monoclonal antibodies can be used in a variety ofapplications. Among these is the neutralization of correspondingproteins. They can also be used to detect viral antigens in biologicalpreparations or in purifying corresponding proteins, glycoproteins, ormixtures thereof, for example when used in an affinity chromatographiccolumns.

The envelope E-glycoprotein polypeptides can be used as antigens toidentify antibodies to West Nile Virus in materials and to determine theconcentration of the antibodies in those materials. Thus, the antigenscan be used for qualitative or quantitative determination of the virusin a material. Such materials include animal tissue and animal cells, aswell as biological fluids, such as animal body fluids, including animalsera. When used as a reagent in an immunoassay for determining thepresence or concentration of the antibodies to West Nile Virus, theantigens provide an assay that is convenient, rapid, sensitive, andspecific.

More particularly, the antigens can be employed for the detection ofmicroorganism pathogen infection (in particular West Nile Virus) bymeans of immunoassays that are well known for use in detecting orquantifying humoral components in fluids. Thus, antigen-antibodyinteractions can be directly observed or determined by secondaryreactions, such as precipitation or agglutination. In addition,immunoelectrophoresis techniques can also be employed. For example, theclassic combination of electrophoresis in agar followed by reaction withanti-serum can be utilized, as well as two-dimensional electrophoresis,rocket electrophoresis, and immunolabeling of polyacrylamide gelpatterns (Western Blot or immunoblot). Other immunoassays in which theantigens of the present invention can be employed include, but are notlimited to, radioimmunoassay, competitive immunoprecipitation assay,enzyme immunoassay, and immunofluorescence assay. It will be understoodthat turbidimetric, calorimetric, and nephelometric techniques can beemployed. An immunoassay based on Western Blot technique is preferred.

Immunoassays can be carried out by immobilizing one of theimmunoreagents on a carrier surface while retaining immunoreactivity ofthe reagent. The reciprocal immunoreagent can be unlabeled or labeled insuch a manner that immunoreactivity is also retained. These techniquesare especially suitable for use in enzyme immunoassays, such as enzymelinked immunosorbent assay (ELISA) and competitive inhibition enzymeimmunoassay (CIEIA).

When either of the immunoreagents are attached to a solid support, thesupport is usually a glass or plastic material. Plastic materials moldedin the form of plates, tubes, beads, or disks are preferred. Examples ofsuitable plastic materials are polystyrene and polyvinyl chloride. Ifthe immunoreagent does not readily bind to the solid support, a carriermaterial can be interposed between the reagent and the support. Examplesof suitable carrier materials are proteins, such as bovine serumalbumin, or chemical reagents, such as glutaraldehyde or urea. Coatingof the solid phase can be carried out using conventional techniques.

The invention provides immunogenic lentiviral vector, and moreparticularly, protective lentiviral vector for eliciting a protectiveresponse against West Nile Virus. These lentiviral vectors can thus beemployed as viral vaccines by administering the lentiviral vector to ananimal susceptible to a pathogenic microorganism (a bacteria, a virus, aparasite) and are useful in preventing infection. In one embodiment, thelentiviral vectors of the invention comprise a heterologous nucleic acidencoding a membrane protein from flavivirus, for example, from West NileVirus, and are useful to prevent infections, for example, from West NileVirus infection. Conventional modes of administration can be employed.For example, administration can be carried out by oral, respiratory,parenteral routes, sublingual route, intranasal route, subcutaneousroute or intradermic route.

The vaccine compositions of the invention are administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective and immunogenic. The quantity to beadministered depends on the subject to be treated including, e.g., thecapacity of the individual's immune system to induce an immune response.

The immunization schedule will depend upon several factors, such as thesusceptibility of the host to infection, the weight of the host, and theage of the host. A single dose of the vaccine of the invention can beadministered to the host or a primary course of immunization can befollowed in which several doses at intervals of time are administered.Subsequent doses used as boosters can be administered as neededfollowing the primary course.

An immunogenic response can be obtained by administering the lentiviralvector of the invention to the host in an amount of about 10 to about500 micrograms antigen per kilogram of body weight, preferably about 50to about 100 micrograms antigen per kilogram of body weight. Theproteins and vaccines of the invention can be administered together witha physiologically acceptable carrier. For example, a diluent, such aswater or a saline solution, can be employed.

To further achieve the objects and in accordance with the purposes ofthe present invention, a kit capable of diagnosing a West Nile Virusinfection is described. This kit, in one embodiment, contains theantibodies of this invention, which are capable of binding to West NileVirus envelope E-glycoprotein. This kit, in another embodiment, containsthe polypeptides of this invention, which are capable of detecting thepresence or absence of antibodies, which bind to the envelopeE-glycoprotein polypeptide.

This invention will be described in greater detail in the followingExamples.

Example 1 Materials and Methods

Cell culture and virus preparations. Human 293T cells and African greenmonkey kidney Vero cells were grown in Dulbecco's modified Eagle medium(DMEM) Glutamax (GIBCO) supplemented with 10% or 5% heat-inactivatedFetal Calf Serum (FCS) respectively. WNV strain IS-98-ST1 (GenBankaccession number AF 481864) [31], a closely related variant of NY99strain [32-34], was propagated in mosquito Aedes pseudoscutellaris AP61cell monolayers. Purification in sucrose gradients, and virus titrationon AP61 cells by focus immunodetection assay (FIA) using anti-WNVHyperimmune Mouse Ascitic Fluid (HMAF) were performed as previouslydescribed [29, 31, 35]. Infectivity titers were expressed as FocusForming Units (FFU).

Lentiviral vector construction and production. The 1.4 kb cDNA codingfor the carboxyl terminal-truncated E glycoprotein lacking thetransmembrane-anchoring region (residues E-1 to E-441; referred to assE_(WNV)) from WNV strain IS-98-ST 1 is described elsewhere [29]. ThesE_(WNV) coding cDNA was already modified by PCR to be flanked on theopen reading frame extremities by BsiWI and BssHII restrictionendonucleases sites. The cDNA was digested with BsiWI and BssHII andthen cloned into the unique BsiWI and BssHII sites of the pTRIP ΔU3 CMVplasmid. The resulting vector plasmid, pTRIP.ΔU3.CMV.sE_(WNV) (hereafterreferred to as pTRIP/sE_(WNV)), contains the IS-98-ST1 sE glycoproteinsequence under the control of the cytomegalovirus immediate earlypromoter (CMVie).

Vector particles were produced by transient calcium phosphateco-transfection of 293T cells with the vector plasmid pTRIP/sE_(WNV), anencapsidation plasmid (p8.7, [36]) and a VSV-G envelope expressionplasmid (pHCMV-G; [37]) as previously described [38]. Quantification ofp24 antigen content of concentrated vector particles was done with acommercial HIV-1 p24 ELISA kit (Perkin Elmer LifeSciences).

Quantitative PCR. For detection of the U5-R sequences in the lentiviralvector, primers and probes used were as follows: probes LTR-FL5′-CACAACAGACGGGCACACACTACTTGA-fluorescein-3′(SEQ ID NO: 1), LTR-LC5′-RED640-CACTCAAGGCAAGCTTTATTGAGGC-phosphorylated-3′ (SEQ ID NO: 2),primers AA55M 5′-GCTAGAGATTTTCCACACTGACTAA-3′ (SEQ ID NO: 3), M6675′-GGCTAACTAGGGAACCCACTG-3′ (SEQ ID NO: 4)[39]. For detection of CD3,the sequences of primers and probes were as follows: Probes CD3-P15′-GGCTGAAGGTTAGGGATACCAATATTCCTGTCTC-fluorescein-3′ (SEQ ID NO: 5),CD3-P2 5′-RED705-CTAGTGATGGGCTCTTCCCTTGAGCCCTTC-phosphorylated-3′ (SEQID NO: 6) and primers CD3-in-F 5′-GGCTATCATTCTTCTTCAAMGGTA-3′(SEQ ID NO:7) and CD3-in-R 5′-CCTCTCTTCAGCCATTTAAGTA-3′ (SEQ ID NO: 8). Primers andprobes were synthesized by Proligo (France). Genomic DNA fromapproximately 3.10⁶ lentiviral vector transduced 293T cells was isolated48 h after transduction using QIAamp® DNA Blood Mini Kit (QIAGEN, GmbH,Hilden). For real-time PCR analysis, 5 μL of DNA were mixed with 15 μLof a PCR master mix consisting of 1× Jumpstart™ Taq ReadyMix™ (Sigma),1.9 mM MgCl₂, 1.5 μM of forward and reverse primers (AA55M/M667 orCD3-in-F/CD3-in-R), 200 nM of the probes (LTR-FL/LTR-LC orCD3-P1/CD3-P2) and, 1.5 units of Taq DNA Polymerase (Invitrogen).Amplifications were performed on a LightCycler 2.0 (Roche AppliedScience), using one cycle of 95° C. for 3 min, and 40 cycles of 95° C.for 5 s, 55° C. for 15 s and 72° C. for 10 s. To take into account thepossible plasmid contamination of vector stocks, DNA from 293T cellstransduced with heat-inactivated (10 min at 70° C.) vector was alwaystested in parallel. For negative controls 5 μL of genomic DNA fromuntransduced cells was used. Each DNA sample was tested in duplicate andthe mean values are reported. Ten-fold serial dilutions of knownconcentration of the plasmid pTripCD3, containing the relevant sequencesU5-R and CD3, were amplified in parallel with DNA samples to generate astandard curve.

The total number of vector copies per cell was calculated by normalizingthe number of U5-R copies to the number of 293T cells, as quantified bythe copy number of CD3 molecules on the same genomic DNA sample, andthen subtracting the number of copies obtained for the heat-inactivatedvector-transduced cells.

Mouse antisera to WNV. Anti-WNV Hyperimmune Mouse Ascitic Fluid (HMAF)was obtained by repeated immunization of adult mice with WNV strainIS-98-ST1 followed by the inoculation of sarcoma 180. The sera to WNVwere obtained by immunization of adult WNV-resistant BALB/c-MBT congenicmice with 10³ FFU of IS-98-ST1 as described previously [31]. Mousepolyclonal anti-WNV antibodies were collected 1 month after inoculation.

Mice immunization and WNV challenge. All animal experiments wereconducted in accordance with the guidelines of the Office Laboratory ofAnimal Care at the Pasteur Institute. Six to eight-week-old 129 micewere intraperitoneally (i.p.) inoculated with varying doses ofTRIP/sE_(WNV) vector particles in 0.1 ml Dulbecco's PBS (DPBS; pH 7.5)supplemented with buffered 0.2% bovine serum albumin (DPBS/0.2% BSA,Sigma). WNV challenge was performed by i.p. inoculation of neurovirulentWNV strain IS-98-ST1 (i.p. LD₅₀=10 FFU) as previously described [29,31]. The challenged mice were monitored daily for signs of morbidity ormortality, for up to 21 days.

Measurement of serum antibody responses. Mice were bled via theperiorbital route and sera were heat-inactivated for 30 min at 56° C.Anti-WNV antibodies were detected by ELISA using sucrose-purified WNVIS-98-ST1 as viral antigen [29, 31]. Peroxidase-conjugated anti-mouseimmunoglobulin (H+L) (Jackson Immuno Research) at a 1:4,000 dilution,and peroxidase-conjugated anti-mouse IgM (μ-chain specific) or IgG(γ-chain specific) (Sigma) at a 1:20,000 dilution were used as secondaryantibodies. The end point titer was calculated as the reciprocal of thelast dilution eliciting twice the optical density (OD) of sera fromTRIP/GFP inoculated mice that served as negative controls.

Anti-WNV neutralizing antibodies were detected by a focus reductionneutralization test (FRNT) on Vero cells as previously described [29].The end-point titer was calculated as the reciprocal or the highestserum dilution tested that reduced the number of FFU by 90% (FRNT₉₀).

Radioimmunoprecipitation assay. Vero cells cultured on 25 cm² flask wereinfected with WNV strain IS-98-ST1 at a high multiplicity of infection.At 20 h post-infection, cells were starved for 1 h in DMEM depleted inmethionine (ICN Biomedicals) and radiolabeled with 100 μCi/mlTrans³⁵S-Label™/ml (ICN Biomedicals) for 3 h. After three washes withcold PBS, cells were lysed with RIPA lysis buffer (50 mM Tris-Cl, 150 mMNaCl, 10 mM EDTA, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, pH 8.0)supplemented with 25 μg/ml aprotinin (Sigma) for 10 min at 4° C. Thecell lysates were then clarified by centrifugation at 10,000 rpm for 5min at 4° C. Radioimmunoprecipitation (RIP) assay was performed asdescribed previously [29, 40]. Viral antigens were immunoprecipitatedwith mouse anti-WNV antibodies. The samples were analyzed by SDS-15%PAGE under non-reducing conditions.

Indirect immunofluorescence and flow cytometry assays. For indirectimmunofluorescence (IF) analysis, human 293T cells cultured on 8-chamberGlass-Labteks (Nunc) were transduced with TRIP/sE_(WNV) vectorparticles. After 48 h, cells were fixed with 3% paraformaldehyde (PFA)in PBS for 20 min and permeabilized with 0.1% Triton X-100 in PBS for 4min. Cells were incubated with anti-WNV HMAF at a 1:100 dilution in PBSfor 1 h. After blocking with DPBS/0.2% BSA, cells were further incubatedwith a Cy3-conjugated anti-mouse IgG antibody (Amersham Pharmacia) at a1:500 dilution in DPBS/0.2% BSA. Cell nuclei were visualized with DAPI.The slides were examined using a Zeiss Axioplan microscope with ApoTomesystem.

For flow cytometry analysis, 293T cells cultured on 25 cm² flasks weretransduced with TRIP/GFP, either heat-inactivated (70° C. for 10 min) ornot. At 48 h, cells were detached, washed and fixed with 2% PFA. The GFPfluorescence intensity was measured by FACS and analyzed with CellQuestsoftware.

Example 2 Expression of the Secreted Form of the E Glycoprotein of WNVby the Recombinant TRIP Lentiviral Vector

To evaluate whether immunization with a lentiviral vector-based vaccinecan protect against WNV encephalitis, a lentiviral vector was generated,TRIP/sE_(WNV), which expresses a soluble form of the E glycoprotein fromthe WNV strain IS-98-ST1 (sE_(WNV)), under the control of the CMVimmediate early promoter (CMV_(i.e.)) (FIG. 1A). We previouslydemonstrated the efficient secretion of sE_(WNV) in the culture mediumof cells infected by a recombinant measles vector [29]. Expression ofsE_(WNV) in lentiviral vector-transduced 293T cells was examined byimmunofluorescence (FIG. 1B). At 48 h post-transduction, a high fractionof cells were immunostained with a pattern suggesting that sE_(WNV)migrated through the secretory pathway.

The amount of physical particles in the vector stock used in this studywas determined by a commercially available ELISA assay against the p24HIV-1 capsid protein (Perkin Elmer Lifescience). The vector stock'sactual titer was calculated on the basis of the transfer of vector DNAto target cells, using a quantitative PCR assay. The quantification ofboth a vector specific sequence (U5) and a cellular locus (CD3) givesthe average vector DNA copy number per cell. This allows a precisetitration of the vector preparation. The TRIP/sE_(WNV) vector stock usedin this study was titrated in human 293T cells at 5.2×10⁷ transductionunits (TU) per ml, (corresponding to approximately 900 TU/ng of p24 inhuman 293T cells). For simplicity reasons, we will express, in thefollowing sections, the quantity of vector particles used as ng of p24antigen.

Example 3 A Single Immunization with TRIP/sE_(WNV) Induces StrongAntibody Responses

To assess the humoral immune response induced by the lentiviral vectorexpressing sE_(WNV), groups of adult 129 mice were inoculated i.p with asingle dose of vector particles equivalent to 500 ng of p24 antigen.Vectors coded either for sE_(WNV) or for GFP protein as a control. Micewere bled periorbitally at 6, 13, 20 or 27 days post-immunization, andindividual or pooled sera were tested for anti-WNV total antibodies, IgGand IgM using an already described isotype specific ELISA [29]. In vitroneutralizing activity of sera was assayed by a focus reductionneutralization test (FNRT) [29].

No anti-WNV antibodies were detected in sera of TRIP/GFP immunizedcontrol mice. Sera from TRIP/sE_(WNV) immunized mice were first testedindividually and very small differences between animals were observed(Mean±SD at day 6=0.5±03; day 13=1.3±0.05; day 21=1.3±0.15; day27=1.4±0.15), thus in following pooled sera were used. In TRIP/sE_(WNV)immunized mice, total antibodies against WNV were detectable as early as6 days post immunization, although present at a low concentration. Asexpected at this time point, only anti-WNV IgM antibodies were detectedin the immune sera. Total antibody responses increased 10-fold to reacha plateau at day 13, and were then maintained over time. At these latertime points (day 13, 20, 27), IgM antibodies disappeared, to be replacedby IgG (Table 1). These sera were reactive with WNV E-glycoprotein fromIS-98-ST1 infected Vero cell lysates as demonstrated by RIP assay (FIG.2A).

TABLE 1 Antibody response of mice to inoculation with TRIP/sE_(WNV)Immunizing WNV WNV IgM vector^(a), antibody antibody WNV IgG Anti-WNVDay of bleeding titer^(b) titer^(b) antibody titer^(b) FRNT₉₀ ^(c)TRIP/GFP Day 27 ND ND ND <10 TRIP/sE_(WNV) Day 06 3,000 300 ND 10 Day 1330,000 ND 1,000 10 Day 20 30,000 ND 1,000 10 Day 27 30,000 ND 1,000 20^(a)Groups of 6 adult 129 mice were inoculated i.p. with a quantity oflentiviral vector particles corresponding to 500 ng of p24 antigen.

A focus reduction neutralization test (FRNT) showed that sera fromTRIP/sE_(WNV) immunized mice contained detectable levels of neutralizinganti-WNV antibodies as early as 6 days post-immunization (Table 1).Together, these data show that an early and specific anti-WNV antibodyimmune response is mounted in mice inoculated with a single dose ofTRIP/sE_(WNV) vector particles.

Example 4 Immunization with TRIP/sE_(WNV) Confers Early Protection ofMice Against WNV Encephalitis

In order to establish if the humoral immunity elicited in mice afterTRIP/sE_(WNV) vaccination was protective, advantage was taken of a mousemodel for WNV challenge. Indeed, some mice strains are extremelysensitive to WNV challenge [31, 41], they develop a neuroinvasive lethaldisease similar to that found in humans [32, 42] and die within few dayspost-inoculation. Groups of 129 mice that received a single dose of 500ng p24 of TRIP/sE_(WNV) or TRIP/GFP vector particles were i.p.challenged with 1,000 i.p.LD₅₀ of WNV strain IS-98-ST1 one or two weeksafter priming.

Remarkably, all mice immunized with TRIP/sE_(WNV) were protected againsta high viral challenge (1,000 i.p. LD₅₀) as early as 7 days afterpriming. All mice immunized with the control vector TRIP/GFP or withDPBS died within 11 days of

TABLE 2 Rapid protection by TRIP/sE_(WNV) against WNV infectionImmunizing vector^(a), Protection^(b) Post-challenge day of challenge(n° of surviving/n° of infected) WNV antibody titer^(c) DPBS Day 7 0/2NA Day 14 0/2 NA TRIP/GFP Day 7 0/2 NA Day 14 0/2 NA TRIP/sE_(WNV) Day 76/6 200,000 Day 14 6/6 300,000 ^(a)Groups of adult 129 mice wereinoculated i.p. with a single dose of lentiviral vector particlescorresponding to 500 ng of p24 antigen or with DPBS.challenge (Table 2). Interestingly, total antibodies against WNVincreased by ten-fold after challenge, suggesting that an effectivesecondary response was mounted in TRIP/sE_(WNV) immunized mice (Table2). Equivalent results were obtained in BALB/c mice (data not shown).These results indicate that TRIP/sE_(WNV) vaccination confers a veryquick, fully protective immune response against a high WNV challenge.

Example 5 Immunization with TRIP/sE_(WNV) Elicits Sterilizing AntiviralImmunity

To address whether or not WNV primo-infection can take place invaccinated animals upon challenge, RIP assays were performed on pooledsera from immunized mice collected before and 21 days after WNVchallenge.

The E protein was first detectable by RIP assay at day 13post-immunization using pooled sera from TRIP/sE_(WNV) immunized mice(FIG. 2A). The lack of detection of antibodies to E in one-week immunesera can be attributed to the low avidity of protein A for IgM in ourRIP assay. Sera from TRIP/GFP control immunized mice did not react withWNV E protein.

Importantly, post-challenge sera from TRIP/sE_(WNV) vaccinated mice didnot precipitate any viral protein other than the E (FIG. 2C). This lackof reactivity against any WNV proteins other than E (such as preM andnon structural proteins NS2a, NS2b and NS3) clearly indicates theabsence of in vivo WNV replication upon challenge in vaccinated animals.In the BALB/c-MBT mice strain, which is resistant to the WNVencephalitis, sera are readily reactive to all viral proteins (Fi 2B).Thus, TRIP/sE_(WNV) vaccination confers full sterilizing immunity tomice.

Example 6 Protection Provided by a Single Injection of TRIP/sE_(WNV) isLong Lasting

We next determined whether a single immunization with the lentiviralvector-based vaccine has the potential to elicit long-term protectiveimmunity against WNV infection. Groups of 129 mice were i.p. immunizedwith TRIP/sE_(WNV) vector particles equivalent to 500 ng of p24 antigenor the same amount of TRIP/GFP vector as a control. Three months later,mice were bled periorbitally and pooled sera from each group were testedby ELISA and FRNT. Antibody levels in TRIP/sE_(WNV) immunized mice werestill remarkably high 3 months after a single vector injection(1:30,000), and neutralizing antibodies persisted (Table 3). Mice werethen challenged i.p. with a 1,000 LD₅₀ dose of IS-98-ST1 WNV. Neithermorbidity nor mortality were observed in mice immunized withTRIP/sE_(WNV) whereas all control mice died within 10 days (Table 3).Total antibody titers as well as neutralizing anti-WNV antibodiesincreased after challenge, suggesting that an effective secondaryresponse was mounted in mice immunized with a TRIP/sE_(WNV) three monthsearlier (Table 3).

Thus, a single dose of the sE_(WNV) coding lentiviral vector isefficient for providing long-term protective immunity in mice.

TABLE 3 Long-term protection by TRIP/sE_(WNV) against WNV infectionAnti-WNV WNV antibody Protection^(d) WNV antibody FRNT₉₀ ^(c) Immunizingtiter^(b) Anti-WNV FRNT₉₀ ^(c) n^(o) of surviving/ titer^(b) (post-vector^(a) (pre-challenge) (pre-challenge) n^(o)of infected(post-challenge) challenge) TRIP/GFP ND <10 0/3 NA NA TRIP/sE_(WNV)30,000 20 13/13 500,000 400 ^(a)Groups of adult 129 mice were immunizedi.p. with lentiviral vector particles corresponding to 500 ng of p24antigen. ^(b)Determined by ELISA on pooled heat-inactivated sera.^(c)FRNT: Focus Reduction Neutralization Test: the highest serumdilution that reduced the number of FFU of WNV by at least 90%. ^(d)Micewere inoculated i.p. with 1,000 LD₅₀ of WNV strain IS-98-ST1, threemonths post immunization. Survival was recorded for 21 days. ND: notdetected (<100) NA: not applicable

Example 7 A Single Minute Dose of TRIP/sE_(WNV) Confers Full and RapidProtection

In order to determine the minimum protective vaccine dose, groups of 129mice were inoculated once with a wide range of TRIP/sE_(WNV) vaccinedoses varying from 1.5 ng to 500 ng of p24 antigen. One week afterpriming, immunized mice were challenged i.p. with 1,000 LD₅₀ IS-98-ST1WNV. As expected, all mice that received 500 ng of p24 of the controlTRIP/GFP vector died within 11-13 days after challenge. A 100%protection was achieved after injection of a single TRIP/sE_(WNV) vectordose as low as 50 ng of p24. Lower doses conferred only partialprotection thus allowing us to calculate a 50% protective dose in adultmice of 6.2 ng of p24 antigen (Table 4). Of note, these dose-protectionexperiments were performed in the most stringent challenge conditions,with an early challenge at day 7 post-vaccination and with a high lethalvirus challenge inoculum (1,000 LD₅₀). Since total antibodyconcentrations increased by ten-fold between days 7 and 14 (Table 1), itis probable that the 50% protective dose would have been even lower than6.2 ng if determined only one week later.

These results demonstrate that a minute dose of vector particlesachieves quick and fully protective immunity in mice. This 100%protection is the consequence of an actual in vivo cell transduction bythe vector particles. A heat treatment (10 min at 70° C.) of the vectorparticles that abrogates the transduction capability of lentiviralvectors (FIG. 3) also abrogates protection (Table 4), ruling out thepossibility of a protection conferred by plasmid DNA or residualsE_(WNV) proteins contaminating the vector stock.

This invention provides the first evidence that lentiviral vectors areefficient tools for eliciting a protective humoral immune responseagainst a pathogen. In addition to the now well-described capacity ofthese vectors for inducing strong cellular immune responses [12-15, 17,18, 21], this work broadens the applicability of lentiviral vectors asvaccination tools against pathogens for which a neutralizing humoralresponse is one active arm of the immune system.

The TRIP/sE_(WNV) vector was able to induce a very early, long-lasting,fully protective immune response against WNV regardless of the lethaldose used in mice. Importantly, this immunity was achieved by a singleimmunization with a minute dose of vector particles.

These features make of TRIP/sE_(WNV) a promising vaccine candidateagainst WNV. In particular, the urgent need for an efficient veterinaryvaccine that could be used in susceptible animal populations in theevent of a WNV outbreak, justifies trials involving the lentiviralTRIP/sE_(WNV) vector in animals, notably horses and birds. Furthermore,the prophylactic immunization of large groups of animals with lentiviralvector-based vaccines would also serve as solid toxicity and safetyproofs of concepts, providing the necessary hindsight and eventuallypaving the way for the possible use of lentiviral vectors asprophylactic vaccination tools in humans.

Several lines of arguments make the TRIP/sE_(WNV) lentiviral vector apowerful candidate vaccine for veterinary use. Firstly, the providedprotection takes place soon after immunization. This could be of majorimportance in the context of a WNV outbreak where rapid protection ofsensitive animals is crucial. The exact nature of this early protectiveimmune response has not been fully addressed. One week afterimmunization IgG antibodies are not yet detectable and IgM antibodiesprobably account for the observed protection. Passive transfer ofpolyclonal anti-WNV IgM has been shown to protect mice from WNVchallenge, demonstrating the critical role of this isotype incontrolling the early phases of WNV infection [43]. Nevertheless, wecannot exclude the contribution of innate or adaptive cellular responsesagainst WNV antigens to the early protection observed [44]. Secondly,the TRIP/sE_(WNV) lentiviral vector is fully effective with a singleminute dose. This makes this candidate vaccine interestinglycost-effective, and could allow the development of protocols for massvaccination, of particular interest for the protection of poultry. It isimportant to note that the dose required for full protective immunitycould have even been overestimated in our mouse experimental protocol.Indeed, it has been shown that murine cells have a lower permissivity tolentiviral vector transduction than other mammal cells, including humancells (data not shown and [4, 45]). Avian cells show a betterpermissivity to transduction than murine cells (data not shown) allowingto predict that minute lentiviral vector vaccine doses would beeffective in fowl. Furthermore, the 50% protective dose of 6.2 ng of p24antigen of TRIP/sE_(WNV) vector particles was calculated under the moststringent challenge conditions (early challenge at day 7post-vaccination and a lethal dose of 1,000 LD₅₀). Given that totalantibody concentrations increase by ten-fold between day 7 and 14, the50% protective dose would probably have been even lower than 6.2 ng ifcalculated only one week later. Thirdly, by virtue of the ubiquitoustropism of the VSV-G envelope used for pseudotyping the vector particles[37], the lentiviral vector vaccine can theoretically be used, with nomodification, in any vertebrate species: horses, fowl and zoo mammals atrisk. A final consideration is that the protective immunity conferred bythe vector is sterilizing, no viral replication takes place after WNVchallenge. This could represent an important advantage if the vaccinewere to be used for bird-immunization. Indeed, while horses, humans andother mammals are believed to be dead-end hosts of WNV infection, birdsare known to be amplifying hosts and participate in the maintenance ofan epidemic [30]. Moreover, recent literature shows that experimentallyWNV infected hamsters that survive the acute illness can continueshedding infectious WNV in urine for up to 52 days [46]. If this can begeneralized to other WNV susceptible mammals, a sterilizing vaccinecould become crucial for the control of the WNV epidemic.

Several WNV vaccines for veterinary use have been proposed. One licensedin the US in 2003 for horses is an inactivated and adjuvanted virus(Fort Dodge website: see equine west nile virus). This inactivatedpreparation elicits low magnitude humoral responses in horses and thusrequires several injections, followed by annual boosts. This killedvirus vaccine does not elicit neutralizing antibodies in ChileanFlamingos and Red-tail hawks [47]. A recombinant canary pox encoding thepreM/E proteins of the WNV has also been licensed in 2004 [48]. Howeverthis candidate also requires two injections at 5 weeks intervals.Neither vaccine provides absolute protection to the vaccinated horses.Other strategies, such as naked DNA encoding pre M and E proteins of WNV[28] are also envisioned.

Candidate vaccines against WNV are for the most part being developed forhuman use. A live measles vaccine expressing the secreted form of theE-glycoprotein of WNV has been shown to efficiently protect mice againstWNV encephalitis [29]. Two recombinant live-attenuated vaccines,chimeric WNV/yellow fever and WNV/Dengue 4, containing the pre M and Egenes of WNV cloned into the backbone of either the 17D vaccine strainof yellow fever virus or the dengue 4 virus, have been tested.Preclinical studies in mice and macaques show that both vaccines areprotective against WNV challenge [49-51]. However, the use of chimericlive-attenuated virus might pose safety concerns: recombination betweendifferent flavivirus species is possible, as demonstrated by naturallyoccurring recombinants flaviviruses [52, 53].

The potential use of lentiviral vector-based vaccines, for human orveterinary use, will require careful design of the vector and rigorouspre-clinical safety studies. Safety concerns about the use ofintegrative vectors are justified in front of the recent reports ofleukemia cases in the SCID. X1 trial. These severe adverse effects aredirectly linked to the integration of the Moloney-derived retroviralvector at close proximity of the LMO2 proto-oncogene in hematopoieticstem cells [54].

However, vaccination applications of lentiviral vectors present lowerrisks. As opposed to stem cell-based gene therapy, which involvesextensive proliferation of transduced cells and persistence of thegenetic modification for the entire life of the patient, transducedcells in a vaccination scenario express the relevant antigen, and thusare targets of the elicited immune response. Cells expressing theantigen, whether APCs or not, are thought to be eliminated from thevaccinated organism within weeks or months. Furthermore, in theparticular case of vaccination by sub-cutaneous or intravenousinjections, it has been shown that VSV-G pseudotyped lentiviral vectorparticles target essentially DCs [13, 18], a non dividing professionalAPC with short in vivo half life upon activation. Nevertheless, bothvector cell tropism and lack of persistence of vaccination vectorsequences, depending on the route of injection and vector dose, willhave to be carefully addressed for each vaccination protocol.

Lentiviral vectors appear to be promising tools for vaccination againstWest Nile Virus and likely other zoonoses or emerging pathogens.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

TABLE 4 Dose-dependent protection by TRIP/sE_(WNV) against WNV infectionImmunizing vector^(a), Protection^(b) Post-challenge dose (ng of p24) n°of surviving/n° of infected WNV antibody titer^(c) TRIP GFP 500 0/6 NAHeat-inactivated TRIP/sE_(WNV) ^(d)  50 0/6 NA TRIP/sE_(WNV) 500 6/6200,000 150 6/6 300,000  50 12/12 300,000  15 5/6 300,000  5 2/5 200,000 1.5  1/12 NA ^(a)Groups of adult 129 mice were inoculated i.p. with asingle dose of lentiviral vector particles. ^(b)Mice were inoculatedi.p. with 1000 LD50 of WNV strain IS-98-ST1 one week after priming.Survival was recorded for 21 days. ^(c)Determined by ELISA on pooledheat-inactivated sera. ^(d)Lentiviral vector particles wereheat-inactivated for 10 min at 70° C. NA: Not Applicable

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1. A lentiviral vector comprising a heterologous nucleic acid encodingan antigen that induces an immunogenic response in vivo, wherein thelentiviral vector is pTRIP ΔU3, and the vector comprises a promoterselected from cytomegalovirus immediate early promoter, CAG promoter,EF1alpha promoter, PGK promoter, SVero promoter, and MND promoter; andwherein the heterologous nucleic acid encodes envelope E-glycoprotein ofa West Nile Virus or a carboxyl terminal-truncated E glycoprotein ofWest Nile Virus lacking the transmembrane anchoring region.
 2. Thelentiviral vector of claim 1, wherein the heterologous nucleic acidencodes envelope E-glycoprotein of West Nile Virus.
 3. The lentiviralvector of claim 1, wherein the heterologous nucleic acid is eitherstrand of a nucleic acid of West Nile Virus encoding envelopeE-glycoprotein.
 4. A host cell transfected or transduced with thelentiviral vector of claim
 1. 5. A lentiviral vector that directs theexpression of a heterologous nucleic acid that encodes an antigen,wherein the vector is pTRIP ΔU3 and the vector comprises a promoterselected from cytomegalovirus immediate early promoter, CAG promoter,EF1alpha promoter, PGK promoter, SVero promoter, and MND promoter; andwherein the heterologous nucleic acid encodes a carboxylterminal-truncated E glycoprotein from West Nile Virus lacking thetransmembrane anchoring region.
 6. A host cell transfected or transducedwith the lentiviral vector of claim
 5. 7. A lentiviral vector comprisinga heterologous nucleic acid encoding an antigen that induces animmunogenic response in vivo, wherein the lentiviral vector is pTRIP ΔU3CMV, and wherein the heterologous nucleic acid encodes envelopeE-glycoprotein of West Nile Virus, or a carboxyl terminal-truncated Eglycoprotein of West Nile Virus lacking the transmembrane anchoringregion.
 8. A host cell transfected or transduced with the lentiviralvector of claim
 7. 9. A lentiviral vector, which comprises plasmidpTRIP/sE_(WNV).
 10. A host cell transfected or transduced with thelentiviral vector of claim
 9. 11. A method of immunizing against apathogenic agent comprising administering a composition as claimed inclaim 1 to an animal in need thereof, wherein the heterologous nucleicacid encodes an antigen, and wherein the antigen elicits antibodiesagainst a pathogenic agent.
 12. The method of claim 11, wherein theanimal in need thereof is selected from humans, horses, birds, poultry,pigs, cattle, rodents, pets, and reptiles.
 13. The method of claim 11,wherein administration of the lentiviral vector elicits long-lastinghumoral immunity against the pathogenic agent.
 14. The method of claim12, wherein the lentiviral vector comprises a nucleic acid encodingcarboxyl terminal-truncated E glycoprotein of West Nile Virus lackingthe transmembrane anchoring region, and wherein the pathogenic agent isWest Nile Virus.
 15. The method of claim 14, wherein the animal is amouse and the humoral response induces protective immunity orsterilizing immunity in the mouse.
 16. The method of claim 15, whereinthe protective immunity is long-lasting.
 17. The method of claim 14,wherein the lentiviral vector is administered by an intraperitonealroute, intravenous route, intramuscular route, oral route, mucosalroute, sublingual route, subcutaneous route, intranasal route, orintradermic route.
 18. The method of claim 17, wherein the lentiviralvector is administered by an intramuscular route, and wherein the animalin need thereof is a horse; or wherein the lentiviral vector isadministered by an intranasal route or an intradermic route, and whereinthe animal in need thereof is a human.
 19. The method of claim 17,wherein the vector is plasmid pTRIP/sE_(WNV).
 20. The method of claim19, wherein the administration of lentiviral vector comprises a dose ofvector particles equivalent to 0.5 ng to 5000 ng of p24 antigen, a doseof vector particles equivalent to 0.5 ng to 50 ng of p24 antigen, or adose of vector particles equivalent to 50 to 500 ng of p24 antigen. 21.The method of claim 20, wherein the lentiviral vector is administeredone time.
 22. A method for the production of envelope E-glycoproteinpolypeptide of West Nile Virus, or a fragment thereof, comprisingculturing a host cell of claim 10 under conditions promoting expression,and recovering the polypeptide from the host cell or the culture medium.